The goals are to understand structure-function relationships in ribosomes and to elucidate mechanisms that govern specific protein-RNA interactions. The research should enhance our ability to understand and control expression of genetic information, which is itself maintained by means of protein-nucleic acid interactions. There are three specific goals. The first is to generate a library of recombinant plasmids; each of which contains a fragment of the gene for 23S rRNA, corresponding to one structural domain. Such restriction fragments of DNA from the plasmid pKK3535, which contains the entire rrnB opreon from Escherichia coli, have been identified. Each fragment will be excised, purified, joined to synthetic DNA linkers and cloned first into pUC18, and then into transcription vectors. These specially constructed plasmids contain promoters for a viral RNA polymerase adjacent to the cloning site. In vitro transcripts, hereafter referred to as domain-RNAs, made from recombinant plasmids will therefore correspond to a structural domain of 23S rRNA and can be used in the second stage of the project to identify 50S proteins that bind to them. Each domain-RNA will be incubated with an unfractionated mix of 50S proteins and ones that remain bound to the RNA following centrifugation through sucrose gradients will be recovered and identified by two dimensional electrophoresis. In the third stage, domain-RNAs will be end-labeled with 32P and subjected to partial chemical modification with appropriate reagents prior to incubation with individual proteins. Nucleotide positions from which modified bases are excluded in protein-bound RNA will be identified by treatment of both free and protein-bound material with aniline and analysis of aniline hydrolysates on sequencing gels. Since the RNA chains are cleaved at modified bases following treatment with aniline, positions where radioactive bands are absent in protein-bound RNA correspond to sites that are required in chemically unaltered form for proper refolding of the RNA or for direct contact with the protein.